Leucine-rich peptide compositions and methods for isolation

ABSTRACT

Disclosed are compositions comprising isolated peptides having a leucine content of from about 12 to about 40 weight percent. Also disclosed is a method for isolating leucine-rich peptides from protein sources such as bovine whey and methods of use for these peptides to provide beneficial effects in a human and/or animal such as increasing blood flow, decreasing blood pressure, increasing muscle mass, improving cognitive function, improving cardiovascular function, etc.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application from U.S. patentapplication Ser. No. 12/411,772, filed Mar. 26, 2009, which claimed thebenefit of priority of earlier-filed U.S. provisional patent applicationNo. 61/039,426, filed Mar. 26, 2008.

FIELD OF THE INVENTION

The invention relates to dietary peptide compositions for stimulatingprotein synthesis, decreasing protein degradation, producingvasodilation, increasing nitric oxide production, and decreasing bloodflow. More specifically, the invention relates to leucine-rich peptidecompositions and methods for their isolation from proteins.

BACKGROUND OF THE INVENTION

Loss of muscle tissue often occurs as a result of aging, malnutrition,and catabolic diseases such as burns, sepsis, and cancer. Dietaryprotein supplementation may be beneficial, but supplementation with theessential amino acid leucine has been shown to be especially beneficial.Dietary leucine has, for example, recently been shown to suppress therate of myofibrillar protein degradation and muscle weight loss in rats.Leucine also stimulates muscle protein synthesis and modulates theactivity of various proteins involved in the control of mRNAtranslation. Leucine may stimulate protein synthesis directly or throughits metabolite, α-ketoisocaproic acid. Leucine may stimulate translationeither independently or by interaction with the mammalian target ofrapamycin (mTOR).

Leucine is one of the branched-chain amino acids and is an essentialamino acid. It is the only amino acid that is converted toacetyl-coenzyme A and alpha-ketoacids and is an important source ofnitrogen for synthesis of glutamine. In addition to its effects onprotein synthesis and degradation, leucine also stimulates glucoseuptake by protein kinase C (PKC), while insulin modulates glucose uptakevia protein kinase B.

Milk-derived whey is one good source of muscle-building proteins. Wheyprotein isolates and whey protein concentrates are used inprotein-building dietary supplements. Whey proteins are a good source ofleucine, but for many individuals who need the muscle-building aminoacids dietary proteins can provide, it is difficult to digest and/orabsorb proteins. It is therefore important to find ways to provideleucine-containing amino acid compositions that provide amino acids in amore bioavailable form for improving muscle protein synthesis.

SUMMARY OF THE INVENTION

The present invention relates to peptides having a leucine content offrom about 12% to about 40% (“leucine-rich peptides”), those peptidesbeing isolated from a protein source by a method comprising the steps ofhydrolyzing the protein in the presence of one or more leucylaminopeptidases, optionally in combination with additional proteases, toproduce a protein hydrolysate, deactivating the enzymes, and filteringthe protein hydrolysate to provide a permeate comprising peptides havinga molecular weight of from about 200 to about 4,000, includingsub-ranges in-between, such as, for example, a molecular weight of fromabout 200 to about 1,000 or a molecular weight of from about 400 toabout 1,000. Optionally, the step of concentrating the leucine-richpeptides by removing the aqueous fluid from the permeate may be added.Also disclosed are method of use of such peptides for a variety ofbeneficial effects, such as producing vasodilation, increasing nitricoxide production, decreasing blood pressure, increasing blood flow,increasing muscle tissue, improving wound healing, and improvingcognitive function.

The invention also relates to a method for isolating leucine-richpeptides from a protein source, the method comprising the steps ofhydrolyzing the protein in the presence of one or more leucylaminopeptidases, optionally in combination with additional proteases, toproduce a protein hydrolysate, deactivating the enzymes, filtering theprotein hydrolysate to provide a permeate, and concentrating thepermeate containing the leucine-rich peptides.

In various embodiments, the protein may be derived from animal or plantsources, including, for example, legumes such as soy or pea, fish, meat,milk, blood, egg, corn, wheat gluten, maize, or combinations thereof. Insome embodiments, the protein source may be bovine milk, bovine whey,whey protein concentrates, and/or whey protein isolates.

In various embodiments, the leucine content of the leucine-rich peptidesmay be within various subranges of the about 12 to about 40 percentrange, such as, for example, from about 12 to about 30 weight percentleucine, from about 15 to about 20 weight percent leucine, from about 15to about 25 weight percent leucine, and from about 20 to about 25 weightpercent leucine.

DETAILED DESCRIPTION

The inventors have discovered that peptides having a leucine content offrom between about 12% and about 40% (“leucine-rich peptides”) may beisolated from a protein source by a method comprising the steps ofhydrolyzing the protein in the presence of one or more (i.e., at leastone) leucyl aminopeptidases, optionally in combination with additionalproteases, to form a protein hydrolysate, deactivating the enzymes,filtering the protein hydrolysate to isolate leucine-rich peptides, andconcentrating the leucine-rich peptides. In one embodiment, the methodcomprises hydrolyzing the protein in the presence of one or moreaminopeptidases which may act as leucyl aminopeptidases (leucineaminopeptidases), optionally supplemented with additional proteases, toform a protein hydrolysate, deactivating the enzymes, filtering theprotein hydrolysate using ultrafiltration, nano-filtration, or anothersuitable filtration method to give a permeate containing theleucine-rich peptides, and concentrating the leucine-rich peptides inthe permeate using reverse osmosis, evaporation, or another suitablemethod for concentrating the peptides by removing the aqueous portion ofthe permeate from the filtration step. The invention also providescompositions comprising leucine-rich peptides prepared by the method.

Dietary leucine appears to have a number of beneficial effects,including stimulation of muscle protein synthesis and suppression of therate of myofibrillar protein degradation and muscle weight loss. Leucinealso increases hypothalamic mTOR signaling and decreases food intake andbody weight (Cota, D. et al. Science (2006) 312: 927-930). Leucineadministration increases leptin levels, and leptin is known to promotelipolysis in adipose tissue, but has no apparent effect on lean tissue.Increased leptin levels result in decreased hunger, decreased foodconsumption, and increased cellular energy expenditure.

Protein sources such as standard whey protein hydrolysates may containas much as 10% leucine, but given the variety of beneficial effects ofthis amino acid and the fact that it is not synthesized in the body fromother amino acids, it has been a goal of the food and nutritionalsupplement industry to develop ways to increase leucine levels in orderto promote its beneficial effects, especially its effects on musclebuilding and inhibition of muscle wasting. The present inventionprovides amino acid compositions that are rich in leucine and suchcompositions may be isolated from readily-available protein sourcesusing the method of the invention.

The inventors proposed to use leucine aminopeptidase to process wheyproteins to produce bioactive/bioavailable leucine-containing peptideswhich they have demonstrated to have a variety of desirable effects.Leucine residues appear to play an important role in the distinctionbetween the effects of remarkably similar proteins. For example,Angiotensin I differs from Angiotensin II by two additionalcarboxyl-terminal amino acids—histidine and leucine. Met-enkephalin, aneuropeptide, has many similar effects to those of Leu-enkephalin, whichappear to be mediated by the aromatic side-chains of the amino acids inthe peptides. Both peptides consist of 5 amino acid residues.Met-enkephalin and Leu-enkephalin differ, however, in thecarboxyl-terminal fifth amino acid residue. In Met-enkephalin, it ismethionine. In Leu-enkephalin it is leucine. Both appear to have effectson arterial blood pressure and vasodilation, but in this area the aminoacid difference appears to be more significant, with Leu-enkephalinappearing to have a stronger effect (Moore, R. H. and D. Dowling,“Effects on Intravenously Administered Leu- or Met-Enkephalin onArterial Blood Pressure,” Regulatory Peptides (1980) 1(2): 77-87). Whileothers have provided hydrolysates containing a significant percentage ofleucine present as a free amino acid (see, for example, U.S. Pat. No.6,875,456 to Delest, V. et al.), the present invention provides smallpeptides (e.g., a hydrolysate containing a significant percentage of di-and tri-peptides) to deliver leucine in a more bioavailable/bioactiveform. These peptides have demonstrated surprising beneficial results,such as potent inhibition of angiotensin converting enzyme (ACE)inhibition, vasodilation, and stimulation of increased nitric oxideproduction. Experiments demonstrate that the peptides of the inventionprovide bioavailable/bioactive leucine, supplementation with thepeptides demonstrating many of the same beneficial effects as thoseprovided by leucine supplementation, but with additional benefits notpreviously described as the result of leucine supplementation.

Leucine-rich peptide compositions of the present invention provideleucine in combination with other amino acids, but provide increasedamounts of leucine in relation to the other amino acids. Thesecompositions may readily be added as a powder to a drink formulation, toa food product, to a nutritional supplement composition such as atablet, capsule, or other formulation, or to a pharmaceuticalpreparation, for example. Additional flavorings, carbohydrates, fats,proteins, vitamins, minerals, and other suitable food or supplementingredients may be included in such compositions.

Peptides of the invention may be used to provide compositions thatprovide a benefit to a human or animal in areas including, but notlimited to, cardiac health, sarcopenia, endothelial function,maintaining healthy blood pressure or lowering blood pressure,vasodilation, muscle growth and development, sports nutrition, infantnutrition, prevention or treatment of metabolic syndrome, cognitivefunction, eye health, diabetes, improvement in glycemic index, mTORactivation, wound healing, and skin care/treatment of skin disorders.

Studies have shown that human athletes given branched-chain amino acids(BCAA) before and during exercise performed better on complex cognitivetests following exercise (Hassmen, P. et al., Nutrition (1994)10:405-10). When tested in dogs, BCAA supplementation appeared to beespecially effective at improving cognitive performance during exercisein older dogs (Fretwell, L. et al. J. Nutr. (2006) 136: 2069S-2071S).Compositions of the present invention provide increased levels of thebranched-chain amino acid (BCAA) leucine, in conjunction with otheramino acids in a form that may be readily incorporated into tablets,capsules, food products such as, for example, nutrition bars,supplements in the form of powders, drink mixes, and other compositionsfor human and/or animal consumption. These compositions may provide abenefit for those individuals who desire to improve their cognitiveperformance. Studies performed with the peptides of the invention havedemonstrated that supplementation with leucine-rich peptides promotesvasodilation and increases blood flow, as well as increasing the rate atwhich glucose is delivered to the tissues. Older individuals, students,and others may derive a particular benefit from foods, drinks, and/orsupplements comprising leucine-rich peptides of the invention.

Metabolic syndrome (Syndrome X) is a chronic disease that affects atleast 1 in every 5 adults in the United States. It is often associatedwith obesity, but the defining characteristic is abnormal glycemiccontrol (glucose intolerance, insulin resistance). Protein-rich diets,especially those comprising significant amounts of leucine, arebeneficial for the treatment of obesity and for the treatment of themetabolic syndrome (Layman, D. K. and D. A. Walker, J. Nutr. (2006) 136:319S-323S). Compositions of the present invention provide an excellentmeans by which the necessary amino acids and the higher amounts ofleucine necessary to affect weight management and the metabolic syndromemay be provided to a human or animal. Products such as “protein water”(water containing whey protein isolate in combination with sweetenersand flavorings), for example, may provide an additional benefit ifpeptides of the present invention are used as the protein component.Snack foods, nutrition bars, drinks, drink mixes, and other products mayalso be formulated with the leucine-rich peptides to providecompositions for use in weight management and loss of fat. Leucine-richpeptide compositions may be used to promote weight loss by incorporatingthose compositions into one or more food products, or may be similarlyused by incorporating them into a nutritional supplement or nutritionaldrink for ingestion at mealtime. Between-meal-snacks may also beformulated with leucine-rich peptide compositions to decrease hunger andfood consumption.

Leucine-rich peptides of the invention may be added to wheycompositions, whey protein isolates, whey protein concentrates, andother protein sources to improve muscle synthesis, decrease musclebreakdown, decrease hunger and food consumption, improve blood flow, andincrease nitric oxide production in the blood vessels, for example.These effects may be beneficial for a large population of individuals,including those who voluntarily wish to build additional muscle mass,those who are losing muscle mass through the process of aging and/orinjury, disease (e.g., sepsis, cancer) or malnutrition, and those whodesire to decrease the percentage of adipose tissue to muscle mass inthe body.

Leucine-rich peptide compositions of the present invention areespecially beneficial for aging individuals or others with sarcopenia.During the aging process and beginning at about age 45, human musclemass decreases. The frailty that is associated with aged individuals maybe attributed in large part to this loss of muscle mass, and thatpredisposes those individuals to further disease or injury. Some of themuscle tissue loss in older individuals may also be the result ofmalnutrition—especially in terms of protein ingestion, as protein-richfoods may tend to be more expensive and less readily available toindividuals on fixed incomes. Compositions of the present inventionprovide nutritional supplements with which to address the needs ofindividuals in whom aging or malnutrition has induced muscle loss.

Body-building involves the combination of nutritional supplementationand exercise to increase muscle mass. Compositions of the presentinvention are ideally suited for body-building nutritional supplements.Leucine-rich compositions stimulate muscle synthesis while generallydecreasing the desire to overeat and gain fat tissue. Furthermore,dietary leucine supplementation has been shown to significantly improveendurance performance (Crowe, M. J. et al., Eur. J. Appl. Physiol.(2006) 97(6): 664-72).

Leucine has demonstrated cardioprotective effects, in addition to itsmany other beneficial effects. For example, leucine protects heartmuscle against myocardial ischemia and some researchers believe it isalso an anti-arrhythmic factor (Gabrys, J. et al. (2002) PharmacologyReviews and Communications 12: 101-108). Peptides of the presentinvention have been shown to act as potent angiotensin converting enzyme(ACE) inhibitors. For example, European Patent Number 1794189B1 (DeSlegte, J et al.) proposes that ACE-inhibitory peptides demonstratingthe greatest efficacy to date are those containing a significantpercentage of proline residues. Those inventors discuss the products ofcompanies such as Calpis and Valio, which provide tripeptides having twoproline residues per tripeptide. Reporting the results of their ownpeptide preparation in terms of ACE inhibition (which is expressed as anIC50 value—the concentration of peptide or hydrolysate needed to reduceACE activity by 50%), they indicate that their hydrolysate produces anIC50 (expressed as μg/ml) of 7.2. Leucine-enriched peptides of thepresent invention, however, have demonstrated IC50 values of from about4.0 μg/ml to about 9.6 μgrams/ml, with an ACE-inhibitory value of from4.0 to 4.7 being the norm for large-scale production lots.

Peptides of the present invention have also demonstrated effectivenessin producing vasodilation, increased blood flow, and increased nitricoxide (NO) production in the blood vessels (see Example 1). Thiscombination of effects clearly demonstrates the positive effects ofpeptides of the invention on the cardiovascular system, and thereforeillustrates the benefits of peptides of the invention ascardio-protective compositions and compositions for improving vascularfunction, increasing blood flow, decreasing blood pressure, increasingendurance capacity, etc. Compositions of the invention may be providedor consumed as dietary supplements and/or pharmaceutical compositionsfor maintaining healthy blood pressure levels and for decreasing bloodpressure in individuals who have or are predisposed to elevated bloodpressure levels. Such compositions may be especially beneficial forindividuals who are diabetic, for example, who may have or bepredisposed to high blood pressure and who may also need the increasedblood flow to tissues in order to decrease tissue damage, especially inthe extremities.

Leucine supplementation has been determined to be more beneficial thanis generic supplementation with BCAAs for burn, trauma, and sepsispatients (DeBandt, J. and L Cynober, J. Nutr. (2006) 136: 308S-313S).Furthermore, leucine has an anabolic effect on proteins in skin woundsand muscle, provided that adequate additional amino acids are available.When compared with leucine supplementation alone, leucinesupplementation in conjunction with protein supplementation provided agreater benefit for wound healing (Zhang, X. et al., J. Nutr. (2004)134: 3313-8). Compositions of the present invention provide peptideshaving enhanced leucine content and are therefore excellent food,pharmaceutical, or nutritional compositions for administration topatients with burns or other skin wounds, muscle injury, trauma, sepsis,and other conditions in which the beneficial effects of leucine may beenhanced by providing leucine in combination with peptides or proteins.These compositions may be provided in oral form, via intravenous means,or by injection. Leucine-rich peptide compositions may also be usedeither orally or topically to enhance skin tone and suppleness, and topromote overall skin health.

Leucine-rich peptides may also provide a benefit for blood pressureregulation and inhibition of angiotensin converting enzyme (ACE). Forindividuals desiring to better manage blood pressure and increase bloodflow to tissues, leucine-rich peptides of the invention may be consumedin food products, drinks and drink mixes, as food supplements such astablets, capsules, powders, and other formulations that may be easilyself-administered at or between mealtimes. By increasing blood flow andpromoting vasodilation and nitric oxide production, compositions of theinvention may provide a benefit by increasing microcirculation,promoting endothelial health, and improving nutrient delivery to thetissues. These effects may be especially important in individualssuffering from diabetes, peripheral artery disease, and other diseasesaffecting circulation, as well as the elderly, athletes, individualsrecovering from surgery, traumatic injury, burns, chronic wounds, andother conditions where increased blood flow and nutrient delivery maypromote improved health.

A variety of beneficial effects have been associated with nitric oxideproduction. NO relaxes smooth muscle and inhibits vascular inflammation,inhibiting endothelial cells lining the blood vessels from releasinginflammatory substances. NO inhibits smooth muscle cell migration andproliferation and decreases platelet adherence and aggregation. Theseeffects may be especially beneficial in those predisposed to thedevelopment of atherosclerosis and to stroke, autoimmune disease, etc.NO appears to regulate vascular inflammation in part by inhibitingexocytosis of Weibel-Palade bodies, which can be triggered by thrombin,histamine, fibrin, complement, leukotrienes, and ATP, for example(Matsushita, K. et al. Cell (2003) 115: 139-150). Leucine-rich peptidesof the present invention have been shown in vivo to increase nitricoxide production and vasodilation. Compositions of the invention maytherefore provide a benefit by preventing the development ofatherosclerotic lesions, decreasing vascular inflammation, decreasingplatelet adherence and aggregation, etc. Compositions of the inventionmay therefore provide supplements that provide a significant benefit instrengthening the cardiovascular system, decreasing inflammation in thecirculatory system, preventing the development of plaques in the bloodvessels, and providing a more “heart-healthy” environment within theblood vessels in general.

The method may be used to isolate leucine-rich peptides from any sourcecomprising leucine-containing proteins. Such sources may include, butnot be limited to, wheat gluten, maize/corn protein isolates, eggproteins, soy and other legume proteins (e.g., pea), fish, meat,blood/blood proteins, and milk proteins. One especially valuable sourceof leucine-rich peptides is milk-derived whey, such as, for example,bovine milk-derived whey, whey protein isolates, and/or whey proteinconcentrates. Whey protein isolates generally have a protein content ofat least about 90 weight percent, while whey protein concentrates mayhave from at least about 35 weight percent to about 80 weight percentprotein.

Enzyme sources for the method of the invention may be readily identifiedby those of skill in the art. Leucyl aminopeptidases (leucineaminopeptidases), for example, are exopeptidases which hydrolyze thepeptide bond adjacent to a free amino group and react most rapidly withleucine-containing peptides and proteins.

Using filtration, molecules are selectively passed through ortransported across a membrane, and this process may be even moreeffective when a pressure gradient is used. Filtration is particularlyeffective at isolating the leucine-rich peptides. Methods of filteringmolecules based upon molecular weight and other properties are known tothose of skill in the art and include membrane filtration generally,ultrafiltration, and nano-filtration, for example. At this point, theisolated leucine-rich peptides are generally contained within apermeate, which is fairly low in solids. The percentage of solids in thecomposition (permeate) may be increased by removal of liquid (aqueousfluid). This may be done by means known to those of skill in the art,including but not limited to processes such as evaporation and reverseosmosis.

Throughout this disclosure, where the term “comprising” is used, it isintended that this term may be substituted with “consisting of” and/or“consisting essentially of,” as well. The invention may be furtherdescribed by means of the following non-limiting examples:

EXAMPLES Example 1 Production of Leucine Peptides

A high protein, low fat, low lactose liquid whey protein isolate productwas used as the beginning substrate. This product was pumped intoreaction tanks at a solids level of 17%. The temperature was raised to45° C. and pH adjusted to 7.3 with sodium hydroxide. A blend ofproteases and aminopeptidases (e.g., leucine aminopeptidase) was thenadded at a level of 0.35% of the solids. Hydrolysis was allowed toproceed for a 6-hour period with hourly pH adjustments to maintain thepH at 7.3. The enzymes were then deactivated by heating the solution to65° C.

Fractionation was performed using filtration, the hydrolysate beingpassed through the filter to retain molecules having a molecular weightof greater than 20,000. The permeate coming from filtration systemcontained the leucine peptides, having approximately 52% of the peptidesin the molecular weight range of less than 1,000; 41% of the peptides inthe molecular weight range of 1,000 to 4,000; and 6% of the peptides inthe molecular weight range of 4,000 to 20,000. These peptides werefurther concentrated using reverse osmosis. The final product was thendried and packaged.

Analysis of six preparations (lots) of peptides of the invention isshown in Table 1, where Lot 06-187, Lot 06-257, and Lot 06-259 aresmall-scale lots and Lot 07-041, Lot 07-046, and 07-055 are large-scaleproduction lots. The degree of hydrolysis of each lot is shown on thebottom row, with a range of hydrolysis of from about 30% to about 50%producing the desired peptides in these experiments. ACE inhibition isshown as the amount of peptide needed to produce 50% ACE inhibition.

TABLE 1 Lot 06-187 Lot 06-257 Lot 06-259 Lot 07-041 Lot 07-046 Lot07-055 Tryptophan 1.52 1.52 1.45 1.34 1.42 1.37 Cystine 0.90 1.48 1.250.63 0.51 0.54 Methionine 3.84 4.47 5.19 5.43 4.95 5.21 Aspartic Acid8.23 8.52 7.76 5.92 5.49 5.63 Threonine 7.14 8.60 8.75 10.77 10.33 11.08Serine 4.31 4.36 4.10 4.78 4.38 4.43 Glutamic Acid 13.43 13.42 12.208.78 8.34 8.47 Proline 4.23 4.79 3.97 2.26 2.28 2.37 Glycine 1.70 1.491.50 1.32 1.19 1.22 Alanine 7.20 6.25 6.53 7.63 7.10 7.08 Valine 7.386.51 6.98 6.87 7.48 7.54 Isoleucine 6.23 6.19 6.38 5.24 6.10 5.87Leucine 14.98 14.80 16.51 22.88 24.45 23.12 Tyrosine 3.44 2.64 2.79 2.552.60 2.38 Phenylalanine 3.61 3.66 3.96 4.11 4.50 4.55 Lvsine, Total 7.987.53 7.12 6.25 5.83 5.98 Histidine 1.81 1.79 1.86 1.73 1.76 1.75Arginine 2.08 1.97 1.72 1.50 1.29 1.39 BCAA 28.6% 27.5% 29.9% 35.0%38.0% 36.5% EAA 58.8% 59.2% 62.2% 67.8% 69.9% 69.4% Protein 79.0 68.769.2 73.1 72.3 72.8 (N = 6.4) Protein 83.7 72.8 73.3 77.5 76.6 77.2 (N =6.78) Calcium 228 306 260 358 392 411 (mg/100 g) Sodium 3374 3829 44754015 2905 2722 Potassium 652 940 949 793 795 622 ACE Inhibition 6.9 9.18.6 4.2 4.4 4.0 (N = 6.4) ACE Inhibition 7.3 9.6 9.1 4.5 4.7 4.2 (N = 6)Degree of 32.8% 35.9% 39.7% 47.3% 47.7% 47.9% Hydrolysis

Examples Example 2 Stimulation of Increased Blood Flow, Vasodilation, NOProduction

Individuals were provided with 2 weeks of daily supplementation witheither the peptide product of the invention, or placebo. Aftercompletion of the first 2 week supplementation period and first day ofvascular testing there was an interval of 1-2 weeks, after which theindividuals started the second 2 week supplementation period, eachindividual consuming the alternative supplement. Individuals wereevaluated at four separate times during the protocol, and asked to fastfor 12 hours, as well as to avoid alcohol, caffeine, and exercise for 24hours, and to consume 36 ounces of water the night before eachevaluation and an additional 12 to 16 ounces of water the morning of thevisit, to ensure that each person was adequately hydrated.

The peptide product was produced by Glanbia Nutritionals, Twin Falls,Id. A single dose of 5 g was premeasured and placed in individualpackets with artificial sweetener. Individuals participating in thestudy were provided with a 2-week supply and instructed to consume onepacket per day mixed in 300 mL water.

On the morning of vascular testing, individuals consumed one packetcontaining 5 g of peptide product or placebo mixed in water. Fastingmeasurements of flow mediated dilation (FMD) and forearm blood flow(FBF) were determined. Fifteen minutes of recovery were allowed betweenFMD and FBF measurements prior to ingestion of the test beverage.Following these baseline measurements, subjects consumed a single 5 gdose of either peptide product or placebo mixed in 300 mL of water andartificial sweetener.

Post-ingestion FMD and FBF measurements were made intermittently. Bloodsamples were obtained at 15, 30, 45, 60, 90, and 120 minutespost-ingestion. Subjects rested in a comfortable position for the entireduration of the test. To ensure standardization between testing trialssubjects were asked to maintain their current level of physical activityduring the study period and to repeat their dietary intake frompreviously recorded diet records the day prior to each vascular testingvisit. Flow mediated dilation (FMD) was assessed using standardizedprocedures for performing high-frequency ultrasonographic imaging before(PRE) and at 30, 60, and 90 min after ingestion of the test beverage.The technique provokes the release of nitric oxide, resulting invasodilation that can be quantitated as an index of vasomotor function.All tests were performed in a quiet, temperature-controlled room after a10 minute period in a supine position. A blood pressure cuff was placedon the upper right arm for occlusion. ECG leads were attached to monitorheart rate throughout the procedure. The brachial artery was imagedabove the antecubital crease, and the transducer was placed to image thebrachial artery in a longitudinal axis with clear visualization of theanterior and posterior vessel walls. When a clear image of the anteriorand posterior walls of the artery was obtained, the transducer was heldby a stereotactic clamp and the position held constant for the durationof the data collection. Baseline brachial artery diameter was recordedfor 30 heart beats. A mark was made on the arm where the image wascollected. The cuff was inflated to 200 mm Hg for 5 min using a rapidcuff inflator (Hokanson E20, Bellevue, Wash., USA) to occlude thebrachial artery, and then released. Arterial diameter was then assessedcontinuously for 300 heart beats after occlusion. Images of the brachialartery were obtained using an Acuson 13.0-MHz linear array transducerand an Aspen cardiac ultrasound system (Acuson Carp, Elmwood Park,N.J.).

Image analysis was performed using MIA software (Medical ImagingApplications, Iowa City, Iowa, USA). For baseline, the average diametertaken from 30 frames was used. Three hundred frames were recorded forpost-occlusion. Peak post-occlusion diameter was calculated by averagingthe vessel diameter 5 frames immediately before the observed peakdiameter and the 5 frames immediately after the same mark. Brachialartery FMD was calculated and expressed as a percentage of the baselinediameter.

Forearm blood flow was measured in the same right arm using venousocclusion strain gauge plethysmography. A calibratedindium/gallium-filled silastic strain gauge, encircled around thelargest diameter of the right forearm, was connected to a plethysmograph(EC6, Hokanson, Inc., Bellevue, Wash., USA). The increase in forearmvolume was measured after blocking the venous efflux by an upper armcuff inflated to 50 mmHg by a rapid cuff inflator (Hokanson E20,Bellevue, Wash., USA) for 7 seconds during each 15-second cycle todetermine resting forearm blood flow (R-FBF). This measurement wasperformed at rest (PRE) and 20, 50, 80, and 110 minutes after ingestionof the test beverage. The hand circulation was excluded by a wrist cuffinflated to 220 mmHg for 1 min before and during each flow evaluation.Forearm blood flow was estimated using specialized software (NoninvasiveVascular Program 3 (NIVP3), Hokanson, Bellevue, Wash., USA) whichdetermined the slope of the change in forearm volume and recorded bloodflow in terms of percent volume change per minute (%/min). Fourplethysmographic measurements were averaged to obtain values for R-FBF.To determine reactive hyperemia induced forearm blood flow (RHFBF), ablood pressure cuff on the upper right arm was inflated to a pressure of200 mmHg for 5 minutes. Upon release of the occlusion, FBF wasdetermined as described above. This measurement was performed at rest(PRE) and 120 min after ingestion of the test beverage.

A blood sample was obtained from a left arm vein after subjects restedquietly for 10 minutes in the supine position. Serum glucose and insulinconcentrations were analyzed in duplicate using a YSI glucose/lactateanalyzer (YSI 2300 STAT, Yellow Springs, Ohio) and commerciallyavailable ELISA [Diagnostic Systems Laboratory (DSL), Webster, Tex.](CV=7.0%) respectively, and used to calculate an index of insulinresistance.

Body mass remained stable over the course of the study, and there wereno significant differences in systolic/diastolic blood pressure after 2weeks of peptide supplementation. However, results demonstrated thatblood vessel diameter, blood flow, and nitric oxide levels wereincreased as a result of ingestion of the peptide product of theinvention.

Example 3 ACE Inhibition Assay

The ACE inhibition assay has been previously described by Cushman andCheung (Cushman, D. W. and Cheung, H. S., Biochem. Pharmacol. (1971) 20:1637). Briefly, substrate was prepared by dissolving 21.475 mg ofHip-His-Leu (“HHL,” Sigma, St. Louis, Mo.) in 8 ml of phosphate bufferedsaline, with volume adjusted to 10 ml and final pH to 8.3. A 10% w/wsolution of peptide composition was prepared using buffer as diluent.Angiotensin-converting enzyme stock solution was prepared by diluting0.1 unit of ACE (rabbit lung, Sigma Chemical, St. Louis, Mo.) withbuffer. To perform the assay, 10 microliters of sample were placed intoa small glass tube with 200 μl of HHL and 70 μl of buffer. Tubes wereplaced in a 37° C. water bath and held for 3 minutes. Tubes were removedfrom the water bath and 20 μl of angiotensin-converting enzyme wereadded to each tube. Tubes were vortexed and returned to the water bathfor 30 minutes. Tubes were then removed from the water bath and 250 μlHCl were added to stop the reaction. Acetic ether (1.5 ml) was added toeach tube with a glass pipette, and tubes were again vortexed. Smalltubes were independently placed into plastic 50 ml tubes to becentrifuged for 2 minutes at 2000 rpm. Tubes were removed from thecentrifuge with care being taken not to disturb the ether layer. Theether layer (1 ml) was removed and placed in a small 10 ml beaker, thenplaced on a hot place to evaporate at approximately 100° C. To eachevaporated sample, 1 ml of nanopure water was added. Absorbance was readat 228 nm with a reaction mixture containing no inhibitor as control.

What is claimed is:
 1. A method for isolating leucine-rich peptides froma leucine-rich protein source, the method comprising the steps of: a)hydrolyzing the protein in the presence of at least one leucylaminopeptidase without additional proteases that are not leucylaminopeptidases to form a protein hydrolysate, b) deactivating theaminopeptidase(s), and c) filtering the protein hydrolysate to produce apermeate containing leucine-rich peptides having a leucine content offrom 12 to 40 weight percent and a molecular weight of from about 200 toabout 4,000 in the permeate.
 2. The method of claim 1 wherein theprotein source is chosen from among the group consisting of legumes,fish, meat, milk, blood, egg, corn, wheat gluten, maize, andcombinations thereof.
 3. The method of claim 1 wherein the step offiltering is performed to collect peptides having a molecular weight offrom about 200 to about 1,000.
 4. The method of claim 1 wherein the stepof filtering is performed to collect peptides having a molecular weightof from about 400 to about 1,000.
 5. The method of claim 1 wherein theprotein source is chosen from among the group consisting of bovine milk,bovine whey, whey protein concentrates, whey protein isolates, andcombinations thereof.